Potentiometric method

ABSTRACT

A potentiometric flow-through electrode detector for use in high performance liquid chromatography and autoanalyzers. Said detector comprises a metal electrode which has the property that it provides a voltage response to the particular molecule to be detected thereby.

This is a continuation of application Ser. No. 249,373, filed Mar. 31, 1981, now abandoned.

The present invention relates to detectors and more particularly to detectors for use in detecting the presence of chemicals.

Present electrode detectors as are used in, say, High Performance Liquid Chromatography (HPLC) or Autoanalyzers are based upon voltammetric methods whereby a current resulting from an applied voltage is measured. Such electrode detectors are complex and subject to interference problems in use. Even more complex and expensive is the use of spectroscopic detectors which are most often supplied in association with present HPLC and autoanalyzers.

The present invention proposes a potentiometric electrode detector for detecting amino acids and other organic copper complexing agents comprising a metallic tube copper electrode adapted to be flow-coupled to the effluent from a liquid chromatograph column or continuous flow analyzer, said electrode and a reference flow-through electrode being adapted for electrical connection to a voltage controller and recording means whereby changes in voltage at the metallic tube electrode due to copper-complexing are recorded. The essential requirement for the metallic tube electrode being such that it must provide a voltage response to the chemical substance to be detected.

In a particularly preferred form of electrode detector the metallic tube electrode is formed from copper and the reference electrode is of platinum and with this form of electrode the following classes of chemicals have been found to be detectable.

Biological Species: Amino acids, proteins, enzymes and enzyme substrates.

Pharmaceuticals: Penicillins, sulphonamides, vitamins.

Complexing Agents: EDTA, ethylenediamine, ammonia.

Oxidising Agents: Hydrogen peroxide, nitric acid.

Metals: All metals capable of reacting with EDTA can be indirectly detected.

The embodiment of a copper tube electrode has advantages which include that it only provides a response to copper-binding molecules and strong redox reagents such that it suffers from fewer background interference problems than present spectroscopic or voltammetric detectors when used for the analysis of complex samples such as blood and urine, etc. Where the electrode detector is miniaturised say having a volume of 0.4 μl, sharp HPLC peaks can be obtained.

The present invention will now be described by way of example with reference to a copper-platinum potentiometric electrode used as a detector for the determination of amino acids by HPLC.

In the drawings:

FIG. 1 is a perspective view of an electrode in accordance with the invention;

FIG. 2 is an exploded view of the electrode of FIG. 1;

FIG. 3 is a schematic diagram of a flow system used in an example of operation of the detector of FIGS. 1 and 2 connected in series with a UV absorbance detector;

FIG. 4 shows a typical chromatogram of a separation of five amino acids using the system of FIG. 3;

FIG. 5 shows calibration plots for the electrode detector system as used in the example; and

FIGS. 6 to 9 relate to plots derived from the example and are described hereinafter.

Referring to FIGS. 1 and 2 there is shown an electrode detector 1 comprised of tubular flow-through components. The detector 1 has a Teflon inlet part comprising a Teflon tubing part 2 fitted into a Teflon mounting part 3. Fitted into part 3 is a platinum reference electrode tube 4 wrapped with a wire contact 5. The other end of platinum electrode 4 is mounted in a further Teflon mounting part 3' which is coupled via Teflon tube 2' to a flow-through copper electrode 6. The exit from copper electrode 6 is through a further Teflon tube 2". In use flow enters the detector from a flow absorbance detector via tube 2 and after passing through detector 1 exits via tube 2".

The following description concerns an example of the use of the detector of this embodiment from which its advantages will be readily appreciated.

EXAMPLE

In this example, we describe the use of a copper tubular electrode as a sensitive and selective detector in the analysis of amino acids by HPLC. A recent paper submitted by P. W. Alexander and C. Maitra to Anal. Chem. entitled "Continuous-flow potentiometric monitoring of α-amino acids with copper wire and tubular electrodes" (not yet published) reports the use of copper wire and copper tubular electrodes as sensitive universal detectors in continuous flow systems, where they were found to be superior to the copper selective membrane electrode for direct quantitation of amino acids. This system is shown to be useful as a detector for amino acids without post-column reaction of the eluted amino acids with Cu²⁺.

Instrumentation

A schematic diagram of the flow system used is shown in FIG. 3. In this system, a Waters model 6000 solvent pump, a Waters Model U6K injector, a Waters Model 450 variable wavelength detector and the electrode detector were linked serially in flow to each other. The electrode detector (FIGS. 1 and 2) consisted of two tubular electrodes, one of copper and one of platinum, both of which were connected to an Activon voltage offset controller (±1.5 V) and the platinum electrode was grounded. The outputs from the UV detector and the voltage controller were linked to a two pen Omniscribe Model B 5271 recorder, which had a 10 mV input.

The components of the electrode detector system were as follows. The internal flow copper tubular electrode was constructed from 0.5 cm diam. copper rod, 2 cm in length. This rod was precisely drilled to give internal diameter of 0.75 mm for the flow path, the length of which was varied over the range 0.5-5.0, mm, however 1 mm was most frequently used. The platinum electrode was a 1 cm length of platinum tubing (0.75 mm ID) sandwiched between two pieces of Teflon tubing which served both to mechanically support the platinum electrode and to provide a means of attaching the flow tubing used to interconnect the various components of the system.

Reagents and Stock Solutions

Amino acids were obtained from various sources: glycine from BDH Biochemicals; valine from Koch Light and Co.; l-isoleucine from BDH Ltd; Methionine from NBC and phenylalanine from Merck. The amino acids were used without further purification and standard solutions were prepared immediately prior to use by dissolving weighed amounts in pH 6.7 buffer. This buffer solution was prepared from AR sodium hydroxide and potassium dihydrogen phosphate (BDH Chemicals).

Volucon standard buffers were used to calibrate the pH meter.

The mobile phase used for the HPLC separation of amino acids was prepared by mixing 50 ml of 1M NaH₂ PO₄, 19.2 ml of 1M NaOH and 10.0 ml of 40% w/v formaldehyde in a 1 l volumetric flask. The solution was then diluted to the mark to give a final pH of 6.7±0.1. All water used for the chromatographic procedure was distilled and passed through a Millipore Q Water Purification System before use and methanol was triply distilled using all glass apparatus.

The urine control sample was obtained from Travenol Laboratories (USA) and was freshly reconstituted prior to use. The pharmaceutical intravenous amino acid solution was also obtained from Travenol, under the trade name "Synthamin 17" and contained 9 essential and 6 non-essential amino acids in concentrations ranging from 400 mg-21 g per liter. This solution was diluted by a factor of 25 for chromatographic runs. Both urine and the intravenous solution were filtered through a 2.5 μm Millipore filter before injection into the chromatograph.

Chromatographic Procedure

Separation by HPLC was accomplished using a μ-Bondapak C18 column (30 cm×3.9 mm I.D., Waters Associates) maintained at 25° C. The column was standardised using the manufacturer's recommended procedure and gave counts in the region of 4000 theoretical plates. The mobile phase flow rate was set at 1 ml/min (unless otherwise stated), producing a back pressure of 1000 psi. The absorbance detector was operated at 200 nm using a sensitivity setting of 0.1 AUFS.

In this study, all eluted compounds were detected both by the UV detector and the copper electrode and the peaks displayed on the two pen recorder using a chart speed of 1 cm/min. Each data point shown in this paper represents the mean of triplicate injections.

To prevent a decrease in sensitivity of the copper electrode due to poisoning, the electrode surface was periodically regenerated by rapid flushing with 5 ml of 8M HNO₃ followed by 20 ml of distilled water and 20 ml of methanol. When the electrode was stored in the flow system, a solvent consisting of 50:50 v/v methanol/water adjusted to pH˜5 was used to prevent deterioration of the electrode.

Results and Discussion

Mobile Phase

The effect of pH on the response of the copper tubular electrode (CTE) was discussed in the previously referenced paper by Alexander and Maitra wherein it was shown that sensitivity for amino acid detection was greatest at high pH values. In this study the optimum pH for the mobile phase was determined to be 6.7 which represented a compromise between atttainment of maximum sensitivity and the prevention of electrode poisoning.

FIG. 4 shows a typical chromatogram of a separation of five amino acids using the CTE as the detector system.

A slightly acidic mobile phase was necessary to discourage the formation of insoluble cupric hydroxides, carbonates and phosphates on the inner surface of the CTE. The ready formation of such compounds in alkaline media would result in potential drift leading to error in the measured concentration.

The purpose of formaldehyde in the mobile phase was to repress negative deviation of the baseline which occurred most prominently prior to the peaks due to sulphur containing amino acids such as methionine and cysteine. The exact mechanism whereby formaldehyde can eliminate such negative baseline deviations is not clear, but its reducing properties may assist in improving the rate of exchange of the amino acids on the electrode surface.

Reference electrode

The platinum tubular electrode shown in FIG. 1 was used for two purposes. Firstly, it acted as an auxiliary electrode to reduce electrical noise and secondly, it served as a reference electrode to the CTE. Since it was electrically grounded, the reference used was the earth potential. This configuration was adopted only after considerable experimentation with conventional reference electrodes, however, we found that the electrically grounded platinum electrode gave greatest sensitivity and least baseline noise.

Electrode flow path

Using glycine as a representative amino acid, the electrode response was studied as a function of the length of the electrode flow path for solutions of three different concentrations of glycine (5.8 μg/ul, 11.5 μg/μl and 17.4 μg/μl). In all cases, the electrode signal reached a maximum for a flow path of 1.0. mm.

Poor response for very short flow paths is considered to be due to the relatively short residence times of the amino acid ligands in the electrode, whereas reduced response in the longer path length electrodes is due to sample dispersion effects. The optimum flow path length of 1 mm was adopted for all further work.

Flow rate

The effect of flow rate on response time and sensitivity was studied by removing the HPLC column, replacing it with a suitable connector and then injecting glycine (3.4 ug/ul) at various flow rates. Values of t_(max), the time required to reach the maximum of the chromatographic peak were measured for each flow rate and the results are shown in Table 1.

TABLE I Response characteristics of the CTE and absorbance detectors at various flow rates.

t_(max) is the time taken to reach the peak maximum

    ______________________________________                                         Flow rate                                                                               Peak height (mm)                                                                               t.sub.max (sec)                                       (ml/min) UV detector CTE     UV detector                                                                              CTE                                     ______________________________________                                         0.5      122         87      24.2      27.0                                    1.0      90          88      13.3      15.8                                    2.0      57          74      6.9       8.5                                     3.0      42          63      4.6       5.5                                     4.0      37          58      3.5       4.0                                     ______________________________________                                    

In agreement with earlier findings, the CTE response improved with increasing flow rate, however this was accompanied by a decrease in sensitivity due to the decreased residence time of the amino acid in the electrode. The response time of the CTE compared favourably with that of the absorbance detector while the sensitivity of the CTE as measured by peak height, was less dependent on flow rate than was the sensitivity of the absorbance detector. It is clear that the optimum flow rate selected would represent a compromise between response and sensitivity; in this study we used a flow rate of 1.0 ml/min.

The relationship between flow rate and resolution was also examined to provide additional information on the response of the CTE. Injections of 1 ul of a solution containing 11.5 and 8.8 μg/ul of glycine and isoleucine, respectively, were made at various values of solvent flow rate and the resultion (R_(s)) of the two resolution peaks was calculated using the conventional formula. For values of R_(s) >0.8, peaks are considered to be only partially resolved, whereas R_(s) >2 indicates resolution with at least two peak basewidths between peak maxima. The results obtained are shown in FIG. 6 using the electrode detector (Δ) and the UV absorbance detector (O).

A decrease in resolution with increasing solvent flow rate was observed for both the absorbance detector and the CTE, however it was observed that resolution achieved with the absorbance detector declined much more rapidly than for the CTE. It is noteworthy however that at all flow rates tested, the UV detector gave slightly superior resolution.

Calibration

Calibration curves for glycine, valine and isoleucine were prepared using both the absorbance detector and the CTE. Linear plots were obtained with the UV detector for the three amino acids in the concentration range 0-25 μg/ul. The calibration plots obtained for the CTE were non-linear and are shown in FIG. 5; the electrode response follows the elution order glycine (A), valine (B), isoleucine (C). The shapes of the CTE calibration plots are similar to those obtained by Loscombe et al. J. Chromatogr. 166 (1978) 403 in their work based on a copper selective membrane electrode.

The precision of the electrode response was estimated by ten replicate injections of a solution containing glycine and isoleucine at concentrations of 1.74 μg/ul and 1.30 μg/ul respectively. Coefficients of variation of 1.4% for glycine and 2.5% for isoleucine were obtained and these results compared favourably with the absorbance detector which gave coefficients of variation of 4.0% and 3.6%, respectively, for the same 10 injections.

Using a definition of detection limit as three times the baseline noise, the calculated detection limits for glycine, valine and isoleucine were 75,200 and 300 ng, respectively, in a 1 μl injection.

We are currently developing a combined recorder offset and signal amplification system which will permit detection of smaller amounts of amino acids than stated above. Initial studies have shown that a ten fold increase in sensitivity over the above values can be easily attained.

Applications

The suitability of the CTE for analysis of urine and a pharmaceutical preparation was investigated. The freshly reconstituted urine sample was not pretreated in any way except for filtration through a Millipore filter prior to injection. The chromatograms obtained for the urine sample using both detectors are shown in FIGS. 7 and 8. These figures also show the chromatograms obtained from urine spiked with five amino acids.

In FIGS. 7 and 8 the dark trace relates to an undiluted urine sample and a spiked urine sample (upper trace) containing the following amounts of amino acids added per ul of urine: (A) glycine (75.μg); (B) valine (5.3 μg); (C) methionine (7.6 μg); (D) isoleucine (4.1 μg); (E) phenylalamine (0.7 μg). Conditions: 1 μl injection with a flow rate of 1 ml/min. FIG. 7 concerns the use of the detector of this embodiment and FIG. 8 the use of a UV absorbance detector (at 200 nm).

The chromatograms obtained using the UV detector are characterised by a profusion of unidentified peaks and this background renders recovery calculations of the added amino acids difficult. In contrast, the CTE chromatogram of the blank urine sample is very "clean" and the peaks produced by the added amino acids in the spiked sample are easily identified. From these chromatograms the recovery percentages for glycine, valine, isoleucine, methionine and phenylalamine were 96%, 106%, 107%, 105% and 100% respectively. The amounts of amino acids added to the spiked sample are shown in the figure. FIG. 7 illustrated a marked advantage of the CTE, that is, its selectivity which eliminates the need for major pretreatment of the sample.

An intravenous solution containing 15 amino acids and excipients such as sodium metabisulphite, sodium acetate and sodium chloride was analysed using the CTE detector. No pretreatment of the sample was involved except dilution and filtration. The chromatogram obtained is shown in FIG. 9. No attempt was made to identify or quantify each amino acid and the separation conditions were not optimised since the purpose was merely to demonstrate the utility of the CTE for pharmaceutical analysis. However, peaks due to 11 of the 15 components can be discerned and it is likely that the remaining amino acids are eluted together with the resolved components.

This example was demonstrated that the CTE can be used as a selective detector in HPLC. In using such an electrode, problems associated with post column derivatisation procedures or absorbance detection of solutes having poor UV absorption are minimised. The performance of the CTE, as indicated by its response time and sensitivity, is comparable to that of the UV absorbance detector. Major advantages of the CTE of this embodiment are: firstly, the selectivity of the CTE is greatly superior to UV detection, and secondly the CTE can be manufactured at very low cost.

In this example, amino acids have been used as solutes to demonstrate the application of the CTE to HPLC. Other copper binding compounds may also be detectable. Narang and Gupta Ind. J. Chem. 13 (1975) 705 have shown that sulpha drugs of sulphanilamide, sulphaguanidine, sulphathiazole, sulphamerazine, sulphadiazine and sulphapyridine readily formed complexes with copper and Evans et al "High Pressure Liquid Chromatography in Clinical Chemistry", Academic Press, London, 1967, P. 71-77, in their deiscussion of the analysis of porphyrins in biological materials by HPLC have reported that capro and meso-porphyrin esters readily complexed copper. We have found that a variety of solutes give response with the CTE.

It will be appreciated that even though the embodiment described has been specific to a copper electrode detector, various metals or metal alloys are suitable for use in accordance with the present invention in relation to the detection of chemicals on the basis that the metal or metal alloy provides a potentiometric response to the molecule to be detected. 

We claim:
 1. A method of detecting amino acids and other organic copper-complexing agents in a fluid, said method comprising the steps of:providing a potentiometric electrode detector comprising a flow-through copper metal tube electrode having a volume in the range of 0.2 to 2 μl and being flow-coupleable to said fluid, said detector including a tubular inlet of material inert to the fluid to be analyzed, a flow-through reference electrode located downstream of said inlet, and an inert flow-through spacer located downstream of said reference electrode, said copper metal tube electrode being located downstream of said spacer, said electrode providing a voltage response to the amino acid or other organic copper-complexing agent to be detected; passing said fluid to be analyzed through said detector; and measuring said voltage response to thereby detect said amino acid or other organic copper-complexing agent.
 2. A method according to claim 1, wherein said reference electrode is of platinum.
 3. A method according to claim 1, wherein said electrode detector is electrically connected to a voltage offset controller which has an output to recorder means.
 4. A method as claimed in claim 1, wherein the volume of the copper metal tube electrode is about 0.4 μl. 